Grain quality in chickpea (<i>Cicer arietinum</i> L.) is a major factor affecting its consumption for human nutrition and health benefits. Some of the major factors affecting chickpea grain quality are: seed weight, size, colour, protein, starch and amylose concentration, and amylopectin structure. The objectives of this study were to: 1) determine variation, repeatability and genotype by environment interaction on thousand seed weight, starch, amylose and protein concentration of chickpea cultivars adapted to western Canada; 2) assess variations in global chickpea germplasm for thousand seed weight, seed size, protein, starch and amylose concentrations; and 3) characterize the desi and kabuli type chickpea for starch concentration, composition, and amylopectin structure to study their effect on starch enzymatic hydrolysis. Limited variation was observed in seed composition of chickpea cultivars adapted to the western Canadian prairies. Significant genotype by environment interaction occurred for starch, amylose, and protein (except for kabuli) concentrations, seed yield and thousand seed weight indicating that testing over a wide range of environments is needed to identify genotypes for grain quality improvement. Repeatability of starch, amylose, and protein concentrations was low and inconsistent across chickpea market classes. Broad sense heritability was higher than repeatability across all traits for all market classes implying that repeatability estimates do not set upper limits to heritability if significant genotype by environment interaction is present. The negative relationship between seed constituents and yield indicates that selection for chickpea cultivars with desired seed composition may require compromise with yield and indirect selection. All the mini core accessions that had above average seed diameter score in both desi and kabuli also had above average score for thousand seed weight. Selecting mini core with promising intrinsic and extrinsic quality characteristics may reduce yield. Slowly digestible starch was negatively correlated with hydrolysis index in both pure starch and meal starch of desi and kabuli. Amylose had a strong relationship with resistant starch but not with rate of starch hydrolysis. Genotypes with a significantly higher rate of starch hydrolysis had significantly lower 60-80 µm starch granule size volume. Amylopectin B2 chains were related to slowly digestible starch of meal (except kabuli) and extracted starch. Resistant starch positively correlated with B1 fraction of amylopectin chain length in both desi and kabuli meal starch. Our results suggest that there is no major difference between starch composition in the two chickpea market classes, although only three genotypes of each class were tested. The meal components affect the starch hydrolytic properties and the effect is genotype specific. The results also show that amylopectin structure influences starch hydrolytic properties. These observations emphasize that complete characterization of seed components is needed to obtain meaningful results regarding the desired nutritional and health benefits attributed to any grain.

This study aims efficient conversion of cotton stalks to cellulosic ethanol through ionic liquid pretreatment and enhanced enzymatic hydrolysis. Among several ionic liquids, EMIMAc exhibited the most striking impact on cotton stalks with respect to the changes in biomass structure and digestibility. Cotton stalks, which were subjected to EMIMAc pretreatment at 10% (w cotton stalks/w EMIMAc) of biomass loading and 150&deg / C for 30 minutes, were found to be 9 times more digestible than untreated cotton stalks. Besides, glucose and ethanol yields, which were based on the cellulose content of untreated cotton stalks, were found as 67% and 66%, respectively. These yields were insufficient regarding efficient conversion of the cellulosic portion of cotton stalks to glucose and ethanol which is linked to the superior solvation capability of EMIMAc towards biomass. In order to enhance aforementioned yields, EMIMAc pretreatment was conducted at 30% of biomass loading. Though lignin extracted was much lower, higher yields were obtained compared to the former case since 96% of cellulose was recovered upon EMIMAc pretreatment and reduced crystallinity was observed for pretreated biomass. Glucose yield was achieved as 84% even at a substrate loading of 15% (w/v). Additionally, 76% of ethanol yield and 3% (v/v) of ethanol titer were obtained upon fermentation. Accordingly, reduction in biomass crystallinity was satisfactory to improve enzymatic accessibility of the biomass. Besides, EMIMAc maintained its effectiveness as a pretreatment agent upon recycling since no change in terms of hydrolysis of pretreated samples was observed upon EMIMAc recycling for three times.

Bioethanol from lignocellulose is expected to be the most likely fuel alternative in the near future. SEKAB E-Technology in Örnsköldsvik, Sweden develops the technology of the 2nd generation ethanol production; to produce ethanol from lignocellulosic raw material. The objective of this master’s thesis was to achieve a better knowledge of the potential and limitations of separate hydrolysis and fermentation (SHF) as a process concept for the 2nd generation ethanol production. The effects of enzyme concentration, temperature and pH on the glucose concentration in the enzymatic hydrolysis were investigated for pretreated spruce at 10% DM using a multiple factor design. Enzyme concentration and temperature showed significant effects on the glucose concentration, while pH had no significant effect on the concentration in the tested interval of pH 4.5-5.5. To obtain the maximum glucose concentration (46.4 g/l) for a residence time of 48 h, the optimal settings within the studied parameter window are a temperature of 45.7⁰C and enzyme concentration of 15 FPU/g substrate. However, a higher enzyme concentration would probably further increase the glucose concentration. If enzymatic hydrolysis should be performed for very short residence times, e.g. 6 h, the temperature should be 48.1⁰C to obtain maximum glucose concentration. The efficiency of the enzymes was inhibited when additional glucose was supplied to the slurry prior to enzymatic hydrolysis. It could be concluded that end product inhibition by glucose occurs and results in a distinct decrease in glucose conversion. No clear conclusions could be drawn according to different techniques for slurry and enzymes, i.e. batch and fed-batch, in the enzymatic hydrolysis process. Investigations of the fermentability of the hydrolysate revealed that the fermentation step in SHF is problematic. Inhibition of the yeast decrease the fermentation efficiency and it is therefore difficult to achieve the 4% ethanol limit. Residence time for enzymatic hydrolysis (48 h) and fermentation (24 h) need to be prolonged to achieve a sufficient SHF process. However, short processing times are a key parameter to an economically viable industrial process and to prolong the residence times should therefore not be seen as a desirable alternative. SHF as a process alternative in an industrial bioethanol plant has both potential and limitations. The main advantage is the possibility to separately optimize the process steps, especially to be able to run the enzymatic hydrolysis at an optimal temperature. Although, it is important to include all the process steps in the optimization work. The fermentation difficulties together with the end product inhibition are two limitations of the SHF process that have to be improved before SHF is a preferable alternative in a large scale bioethanol plant.

Mechanical pressing of soybean is highly desirable as it provides, at low cost, non-contaminated, protein-rich, low-fat soyflour which can be further processed into nutritious edible foods. Unfortunately, mechanical pressing of this low-fat oilseed ($<$20%) yields only 50-70% of the available oil, in contrast to the solvent extraction method which recovers over 98% of the oil. The main focus of the study was to maximize the oil recovery from soybean using mechanical oil expression by applying two pretreatments, enzymatic hydrolysis and extrusion cooking of soybeans, and by varying the pressing conditions including three applied pressures (20, 40 and 60 MPa), three pressing temperatures (22, 60 and 90°C) and two sample sizes (10 and 20 g). To characterize the material properties affecting mechanical oil expression from soybean a mathematical simulation of uniaxial compression was developed which incorporated the time dependent variation of soybean properties. The mathematical simulation was based on Terznaghi's theory of consolidation for soils and was solved using measured values of the coefficients of permeability, volume change and consolidation. A compression-permeability test cell was specifically developed for these measurements. For validation of the model, in addition to extruded soy, sunflower seeds (oil content ca. 45%) were also compressed under the same pressing conditions. Improvements in oil recovery due to enzymatic pretreatment of soybean were small, while the extrusion pretreatment increased the oil recovery from only a trace for raw soybean to 90.6%. Such oil recovery using mechanical pressing of soybean has not been reported in the past. The measured values of oil recovery, coefficients of permeability, volume change and consolidation for soybean and sunflower seeds were found to vary significantly $(P<0.05)$ with time of pressing, applied pressure, pressing temperature and the size of the sample. For extruded soy samples, the developed model predicted the values of oil recovery versus pressing time with an average error of 15%, while for sunflower seed samples the average prediction error was 40%. The high error values were attributed to the presence of hulls in the sunflower seed samples, as well as error during measurement of the coefficient of permeability. The coefficient of consolidation was found to have the greatest influence on oil recovery. The incorporation of time dependent material properties in the developed simulation was demonstrated to give more accurate and consistent prediction in trends of oil recovery as compared to using constant material properties. The correlationship developed between the oilseed material properties and the oil recovery obtained from uniaxially compressed oilseeds would help researchers and designers to better evaluate the mechanical oil expression equipment and systems. To the extent that the developed model adequately predicted oil recoveries from both sunflower and soybean oilseeds, the model is expected to be applicable to other oilseeds as well.

Grain quality in chickpea (<i>Cicer arietinum</i> L.) is a major factor affecting its consumption for human nutrition and health benefits. Some of the major factors affecting chickpea grain quality are: seed weight, size, colour, protein, starch and amylose concentration, and amylopectin structure. The objectives of this study were to: 1) determine variation, repeatability and genotype by environment interaction on thousand seed weight, starch, amylose and protein concentration of chickpea cultivars adapted to western Canada; 2) assess variations in global chickpea germplasm for thousand seed weight, seed size, protein, starch and amylose concentrations; and 3) characterize the desi and kabuli type chickpea for starch concentration, composition, and amylopectin structure to study their effect on starch enzymatic hydrolysis. Limited variation was observed in seed composition of chickpea cultivars adapted to the western Canadian prairies. Significant genotype by environment interaction occurred for starch, amylose, and protein (except for kabuli) concentrations, seed yield and thousand seed weight indicating that testing over a wide range of environments is needed to identify genotypes for grain quality improvement. Repeatability of starch, amylose, and protein concentrations was low and inconsistent across chickpea market classes. Broad sense heritability was higher than repeatability across all traits for all market classes implying that repeatability estimates do not set upper limits to heritability if significant genotype by environment interaction is present. The negative relationship between seed constituents and yield indicates that selection for chickpea cultivars with desired seed composition may require compromise with yield and indirect selection. All the mini core accessions that had above average seed diameter score in both desi and kabuli also had above average score for thousand seed weight. Selecting mini core with promising intrinsic and extrinsic quality characteristics may reduce yield. Slowly digestible starch was negatively correlated with hydrolysis index in both pure starch and meal starch of desi and kabuli. Amylose had a strong relationship with resistant starch but not with rate of starch hydrolysis. Genotypes with a significantly higher rate of starch hydrolysis had significantly lower 60-80 µm starch granule size volume. Amylopectin B2 chains were related to slowly digestible starch of meal (except kabuli) and extracted starch. Resistant starch positively correlated with B1 fraction of amylopectin chain length in both desi and kabuli meal starch. Our results suggest that there is no major difference between starch composition in the two chickpea market classes, although only three genotypes of each class were tested. The meal components affect the starch hydrolytic properties and the effect is genotype specific. The results also show that amylopectin structure influences starch hydrolytic properties. These observations emphasize that complete characterization of seed components is needed to obtain meaningful results regarding the desired nutritional and health benefits attributed to any grain.

ABSTRACT STEREOSELECTIVE SYNTHESIS OF OPTICALLY ACTIVE CYCLITOL PRECURSORS VIA CHEMOENZYMATIC METHOD AND SYNTHESIS OF A NUCLEOSIDE PRECURSOR Oguzkaya, Funda M.S., Department of Chemistry Supervisor: Prof. Dr. Cihangir Tanyeli June 2006, 99 pages &amp / #945 / &#039 / -acetoxylation of &amp / #945 / ,&szlig / -unsaturated cyclic ketones was adjusted via Mn(OAc)3 in regioselective manner. Then, PLE hydrolysis was carried out so as to afford enantiomerically enriched &amp / #945 / &#039 / -acetoxylated and &amp / #945 / &#039 / -hydroxylated cyclic compounds. From our knowledge about the literature and previous works dealing with &amp / #945 / &#039 / -hydroxylated products which are easily racemized, protection was directly adjusted via acetylation so as to prevent this possibility. Resulting enantiomerically enriched products were subjected to Upjohn Dihydroxylation to obtain cyclitol precursors and following Luche Reduction of ketone was adjusted so as to obtain corresponding cyclitols. In addition with such synthetic design, firstly dimethyl cyclopent-3-ene-1,1-dicarboxylate was obtained so as to reach in former manner 3-cyclopentene-1,1-dicarboxylic acid, and in latter manner cyclopent-3-enecarboxylic acid. Resulting compound was converted to 6-iodo-2-oxa-bicyclo[2.2.1]heptan-3-one and followingly to the target nucleoside precursor which is 2-oxa-bicyclo[2.2.1]hept-5-en-3-one.

Chiral polyfunctionalized 1,5-dicarbonyl compounds are important synthetic intermediates and starting materials for many biologically active compounds so their synthesis has a great importance in the literature. In the first step, 1,3-cyclohexandione and other b-diketone derivatives are protected under acid catalyzation and their corresponding b-keto enol ether derivatives are obtained. These b-keto enol ethers are then converted to a-acetoxy enones in racemic form by Mn(OAc)3 mediated oxidation. Enzymatic kinetic resolution is applied to the racemic acetoxy enones by using different lipases and enantiomerically pure a-acetoxy and hydroxy enones are obtained. Then, dicarbonyl derivatives are obtained by hydrolizing racemic a-acetoxy enones. Oxidative cleavage of racemic a-acetoxy diketones in the presence of oxone gives corresponding racemic 1,5-dimethyl ester derivatives. By using this reaction as a reference, same reactions are applied to the chiral a-acetoxy and hydroxy diketones in order to synthesize chiral a-acetoxy and hydroxy 1,5-diester derivatives.

Lignocellulosic biomass is under extensive investigation as a bioethanol and bio-based materials feedstock. However, the complex structural and chemical mechanisms of lignocellulosic plant, which cause resistance to deconstruction during saccharification, require a pretreatment process. In this study, raw materials (corn bran, wheat bran and wheat straw) were selected because of their production and consumption in Turkey and also their accessibilities to be used as bioethanol source. Microfluidization pretreatment (high-pressure fluidization), which stands as a new approach for nano-cellulosic fibers production, was studied at 500 bar and 2000 bar to observe the qualitative and quantitative modifications in enzymatic hydrolysis depending on its effects on lignocellulosic structure. Optimum cellulase concentrations were determined for microfluidized samples as 4.5 U/g dry biomass for wheat bran, corn bran and 6.0 U/g dry biomass for wheat straw samples for the first 150 min interval. Effective usage of solid loads were found as 5.0 %, 2.5 %, and 7.5 % (dw/v) for wheat bran, wheat straw and corn bran, respectively. X-ray diffraction and SEM results of the microfluidized samples have indicated that the pretreatment has increased crystallinity index of all the samples and resulted in a scattered structure. Comparisons with other methods (softening, dilute-acid and lime pretreatments) have shown that microfluidization is advantageous over others by reducing the time required for enzymatic hydrolysis and thus can be a promising alternative pretreatment.

Food protein-derived peptides have increasingly become important sources of ingredients for the formulation of therapeutic products. The main aim of this work was to study the in vitro and in vivo bioactive properties of structurally diverse group of peptides produced through enzymatic hydrolysis of flaxseed proteins (FP). Hydrolysis of FP with seven proteases followed by fractionation into low-molecular-weight (LMW) cationic fractions yielded multifunctional peptides that inhibited angiotensin converting enzyme (ACE) and renin activities, which are molecular targets for antihypertensive agents. The LMW peptides also exhibited antioxidant properties by scavenging free radicals and inhibiting amine oxidase activity. The peptide fractions showed inhibition of calmodulin-dependent phosphodiesterase, an enzyme that has been implicated in the pathogenesis of several chronic diseases. Moreover, FP hydrolysis with thermolysin and pronase followed by mixing with activated carbon yielded branched-chain amino acids (BCAA)-enriched multifunctional peptide mixture (Fischer ratio of 23.65) with antioxidant properties and in vitro ACE inhibition; Fischer ratio of 20.0 is considered minimum for therapeutic purposes. The BCAA-enriched peptide product can be used in clinical nutrition to treat muscle wasting symptoms associated with hepatic diseases. Furthermore, an arginine-rich peptide mixture (31% arginine versus 11% in the original flaxseed protein) was produced by hydrolysis of FP with trypsin and pronase followed by separation using electrodialysis-ultrafiltration. Arginine plays important physiological roles especially as precursor to vasodilator, nitric oxide. The arginine-rich peptide mixture exhibited in vitro ACE and renin inhibition and led to decreased systolic blood pressure (–17.9 and –11.7 mmHg, respectively at 2 and 4 h) after oral administration to spontaneously hypertensive rats. For the first time in the literature, we showed that arginine peptides have superior physiological effects when compared to the amino acid form of arginine. Lastly, quantitative structure-activity relationship studies using partial least squares (PLS) regression yielded two predictive models for renin-inhibiting dipeptides with z-scales amino acid descriptors. The PLS models indicated that hydrophobic and bulky side chain-containing amino acids contribute to renin inhibition if present at the amino- and carboxyl-terminal of dipeptides, respectively. Based on this study, Ile-Trp was discovered as potent renin-inhibiting dipeptide, and may serve as a useful template for the development of potent antihypertensive peptidomimetics.

In recent years, fungal and bacterial cellulases have gained popularity for the conversion of lignocellulosic material to biofuels and biochemicals. This study investigated properties of fungal (Trichoderma. reesei) and bacterial (Clostridium thermocellum) cellulases. Enzymatic hydrolysis was carried out with T. reesei using nine enzyme concentration and substrate combinations. Initial rates and extents of hydrolysis were determined from the progress curve of each combination. Inhibition occurred at the higher enzyme concentrations and higher solids concentrations. Mechanisms to explain the observed inhibition are discussed. Samples of C. thermocellum purified free cellulase after 98% hydrolysis were assayed to determine the total protein content (0.15 ± 0.08 mg/mL), the enzymatic activity (0.306 ± 0.173 IU/mL) and the cellulosome mass using the Peterson method for protein determination, the cellulase activity assay with phenol-sulfuric acid assay, and the indirect ELISA adapted for C. thermocellum cellulosomes, respectively. Issues regarding reproducibility and validity of these assays are discussed.